Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “doublet detection”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 379 records · Page 21Linked to original sources
PubMed1986

Biochemical characterization of the murine activated lymphocyte alloantigen Ly-6E.1 controlled by the Ly-6 locus.

The Ly-6.1 alloantigen defined by monoclonal antibody SK70.94 and expressed predominantly on activated lymphocytes has been immunoprecipitated from lysates of cells biosynthetically radiolabeled with 35S-methionine. On SDS-polyacrylamide gel electrophoresis the reduced antigen appeared as a doublet of 17 and 18 kD. The nonreduced polypeptides had higher mobilities indicating intrachain but not interchain disulfide linkages. The nonreduced form was detectable by immunoperoxidase stain on blots after SDS-PAGE. This showed both polypeptide species contained the antigenic epitope. Labeling in the presence of tunicamycin did not change the apparent m.w. suggesting the absence of N-linked carbohydrate. Pulse-chase data are inconsistent with a strict precursor-product relationship between the two polypeptides and give a half-life for the antigen in 2-day Con A blast cells of about 4 1/2 hr. A highly purified preparation of this antigen displayed a similar electrophoretic pattern and, in the presence of deoxycholate, eluted from a Sephacryl S-200 gel filtration column with a partition coefficient equivalent to that of a 31-kD soluble globular protein. Because of the associated detergent and probable deviation from globularity, this is an over-estimate of the size of the native molecule, and is more consistent with the native molecule being a single polypeptide chain rather than a dimer. Isoelectric focusing of this material showed microheterogeneity with at least five bands between pI 4 and 5. Another monoclonal antibody, HD-42, which had been isolated on the basis of its specificity for a fibrosarcoma antigen subsequently found to be Ly-6 related, precipitated the same polypeptides. Furthermore, no obvious difference was evident between precipitates from Con A-activated lymphocytes and Meth A fibrosarcoma cells.

Animals↗
PubMed1981

Purification and characterization of human coagulation factor V.

We have purified human coagulation Factor V 6,000-fold to homogeneity from citrated plasma using polyethylene glycol 6000 precipitation, adsorption of Factor V to barium citrate, DEAE-Sepharose chromatography, and gel filtration on Ultrogel AcA 34 (yield 21%). Human Factor V is a single polypeptide chain before and after disulfide bond reduction with an apparent Mr = 335,000 as determined by electrophoresis on 5% acrylamide sodium dodecyl sulfate gels. Human Factor V is a glycoprotein containing 13% of weight carbohydrate and there is a high content of sialic acid (86 residues/mol) compared to the other sugars. When human Factor V is treated with thrombin, coagulation activity increases 25- to 30-fold to a specific activity of 1.7 to 2.0 units/microgram. Thrombin activation is accompanied by the cleavage of three bonds in the Factor V molecule. We have detected activation intermediates with apparent Mr = 295,000 and 248,000 and final products with apparent Mr = 150,000, 121,000, and a doublet at 95,000-91,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final products of thrombin activation of human Factor V and bovine Factor V are similar, yet the intermediates observed are different. This suggests that cleavages are made at similar locations in bovine and human Factor V, but that they occur in a different sequence. When human Factor V is treated with the Factor V activator from Russell's viper venom, it is split into two components with apparent Mr = 303,000 and 95,000-91,000 and is fully activated. The increase in coagulation activity observed upon treatment of human Factor V with thrombin or the Factor V activator from Russell's viper venom seems to correlate with the generation of the doublet Mr = 95,0090-91,000 component.

Amino Acids↗
PubMed1998

Tektins as structural determinants in basal bodies.

Tektins, present as three equimolar 47-55 kDa protein components, form highly insoluble protofilaments that are integral to the junctional region of outer doublet microtubules in cilia and flagella. To identify and quantify tektins in other compound microtubules such as centrioles or basal bodies, a rabbit antiserum was raised against tektin filaments isolated from Spisula solidissima (surf clam) sperm flagellar outer doublets and affinity-purified with nitrocellulose blot strips of tektins resolved by SDS- or SDS-urea-PAGE. These antibodies recognized analogous tektins in axonemes of organisms ranging from ctenophores to higher vertebrates. Quantitative immunoblotting established that outer doublet tektins occur in a 1:17 weight ratio to tubulin. Cilia and basal apparatuses were prepared from scallop gill epithelial cells; cilia and deciliated cells were prepared from rabbit trachea. Tektins were detected by immunoblotting in basal body-enriched preparations while tektins were localized to individual basal bodies by immunofluorescence. Supported by greater fluorescence in basal bodies than in adjacent axonemes in tracheal cells, analysis of basal apparatuses demonstrated both a proportionately greater ratio of tektin to tubulin (approximately 1:13) and two distinct solubility classes of tektins, consistent with tektins comprising the B-C junction of triplets in addition to the A-B junction as in doublets.

Animals↗
PubMed1986

Correlation of treponemicidal activity in normal human serum with the presence of IgG antibody directed against polypeptides of Treponema phagedenis biotype Reiter and Treponema pallidum, Nichols strain.

Normal human serum (NHS) was shown to have complement-dependent treponemicidal activity against both Treponema pallidum and Treponema phagedenis biotype Reiter (TPR) by employing in vitro-in vivo neutralization and TPR plaque assays, respectively. The molecular basis of NHS treponemicidal activity was studied by immunoblot analysis in conjunction with treponemicidal assays. Five major T. pallidum polypeptide bands (47kDa, 35kDa, 33kDa doublet, and 30 kDa) and three major TPR polypeptide bands (47kDa and 33kDa doublet) bound IgG present in NHS. Absorption of NHS with TPR completely removed both TPR and T. pallidum treponemicidal activity; corresponding immunoblots demonstrated a significant removal of IgG antibody against all three TPR polypeptide bands as well as four T. pallidum polypeptide bands (30kDa, 33kDa doublet, and 35kDa). In contrast, T. pallidum absorption of NHS was found to remove treponemicidal activity against T. pallidum but not TPR; corresponding Western blots showed the complete removal of IgG antibody against all but one T. pallidum polypeptide band (47kDa) but no detectable loss in IgG antibody against the TPR polypeptides. These results suggest that antibody in NHS generated against nonpathogenic, indigenous treponemes is responsible for the T. pallidum treponemicidal activity. Furthermore, the treponemicidal activity against T. pallidum correlated with the presence of IgG antibody against T. pallidum polypeptides of 30kDa, 35kDa, and a 33kDa doublet.

Antibodies, Bacterial↗
PubMed1992

Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast.

Two proteins of 145 and 143 kDa were identified in pea which co-purify with a chloroplast processing activity that cleaves the precursor for the major light-harvesting chlorophyll binding protein (preLHCP). Antiserum generated against the 145/143 kDa doublet recognizes only these two polypeptides in a chloroplast soluble extract. In immunodepletion experiments the antiserum removed the doublet, and there was a concomitant loss of cleavage of preLHCP as well as of precursors for the small subunit of Rubisco and the acyl carrier protein. The 145 and 143 kDa proteins co-eluted in parallel with the peak of processing activity during all fractionation procedures, but they were not detectable as a homo- or heterodimeric complex. The 145 and 143 kDa proteins were used separately to affinity purify immunoglobulins; each preparation recognized both polypeptides, indicating that they are antigenically related. Wheat chloroplasts contain a soluble species similar in size to the 145/143 kDa doublet.

Biological Transport↗
PubMed1996

Differential expression of proteins in muscle and electric organ, a muscle derivative.

The electric organ of electric fish develops from a myogenic lineage. We have used immunohistochemistry and immunoblotting to determine which features of the muscle phenotype are retained and whether any new ones are expressed in mature electrocytes of the electric fish Sternopygus. The muscle-specific intermediate filament desmin was found throughout the electrocytes, and different desmin antibodies detected molecules with different subcellular distributions. Western blots confirm that these antibodies recognize a protein of MW = 53 kD, the molecular weight of desmin. Other muscle proteins were also present within electrocytes: Actin and sarcomeric alpha-actinin were found within the subsynaptic membrane beneath the plasmalemma of the electrocytes, and talin and acetylcholine receptors were detected both at the innervated posterior face and at the non-innervated anterior face. This was confirmed using rhodamine-conjugated alpha-bungarotoxin. Neither myosin heavy chain nor tropomyosin was present in electrocytes. Finally, we detected within electrocytes a type I acidic keratin that forms a filamentous meshwork within each cell. Immunoblots corroborate this result: A keratin-positive doublet of MW = 50 kD and 57 kD was found in both electrocytes and skin. Myosin, actin, talin, tropomyosin, desmin, alpha-actinin, and acetylcholine receptor, but not keratin, were all expressed in fish skeletal muscle fibers. Thus, electrocytes retain some muscle-specific proteins, do not express others, and in addition, express a non-muscle protein.

Actinin↗
PubMed1994

Hunt for singlet oxygen under in vivo conditions.

It is widely accepted that applying exogenous photosensitizers in living organism the photodynamic effect is mediated by singlet oxygen formed by energy transfer from excited sensitizer to oxygen present in the cells (Type II mechanism). Though in homogeneous solutions singlet oxygen was detected during sensitization, efforts to observe it in vivo failed, so far. Kinetic considerations based on the assumption that triplet sensitizer molecules interact with (doublet) free radicals accumulated in the tissue as well as on literature data indicate that the steady state concentration of singlet oxygen can decrease by more than one order of magnitude at transition from homogeneous systems to in vivo conditions making it undetectable.

Kinetics↗
PubMed2001

Evidence that the chromogranin B fragment 368-417 extracted from a pheochromocytoma is phosphorylated.

A rabbit antiserum was raised against a synthetic peptide corresponding to residues 403 to 417 of human chromogranin B. This peptide was chosen to match the potential C-terminal end of a putative proteolytic fragment of the protein located between dibasic doublets in positions 366-367 and in positions 418-419 of the precursor. A radioimmunoassay based on this antiserum was developed and used to detect the protein or a fragment thereof in a pheochromocytoma tumor extract. One fragment was purified to homogeneity by successive reverse-phase HPLC chromatographies. The N-terminal sequence established by automated Edman degradation, was N-Y-P-S-L-E-L-D-K-M-A-H-G-Y-G-E-E-S-E-E-E-R corresponding to the 368-389 sequence of human chromogranin B. Taking into account the specificity of the antiserum used for peptide identification and alignment with the precursor sequence, we deduced that the purified peptide was chromogranin B (368-417) and represented a new peptide generated by limited proteolysis of chromogranin B. Combining electrospray mass-spectrometry and enzymatic dephosphorylation, we demonstrated that this peptide was phosphorylated.

Adrenal Gland Neoplasms↗
PubMed1995

Human proteasomes analysed with monoclonal antibodies.

The proteasome or multicatalytic endopeptidase from eukaryotic cells consists of at least 14 subunits that fall into two families, alpha and beta. Subunit-specific monoclonal antibodies against ten different subunits of human proteasomes have been produced, together with an antibody that reacts with a motif (prosbox 1), common to alpha-type subunits. Four of the subunit-specific antibodies were able to precipitate proteasomes. The subunit composition of HeLa-cell proteasomes precipitated with these four different antibodies were identical, as judged from two-dimensional electrophoresis. One of the four antibodies was used to obtain proteasomes from cell lines (HeLa, Daudi, IMR90 and BSC-1) and human tissues (placenta, kidney, and liver). Electrophoretic analysis of these proteasomes, combined with peptide mapping of some subunits, suggests that they all contain 14 types of subunits as their major constituents. However, one subunit was present in two isoelectric isoforms in all cells examined. Two other subunits occurred in two or three isoelectric isoforms in placenta, liver and kidney, but not in the cell cultures. Extracts of human cells (HeLa, IMR90, Daudi and erythrocytes) were analysed by non-denaturing electrophoresis and immunoblotting. All of the 11 subunits detected by antibodies were present in a pair of ATP-stabilized protein complexes, presumed to be the 26 S proteinase, and in a doublet of complexes which migrated more slowly than purified proteasomes. Besides being present in proteasomes, one subunit was also found to occur in the free state in cell extracts.

Amino Acid Sequence↗
PubMed1998

Collagenase-1 complexes with alpha2-macroglobulin in the acute and chronic wound environments.

The purpose of this study was to examine the appearance and activation of collagenase-1 (MMP-1) in the wound environment. We found that MMP-1 accumulates in the fluid phase of the burn wound environment within 2 d of injury and reaches maximal levels by day 4. Two forms of the enzyme were evident; one that corresponded to proMMP-1 and another that corresponded to a group of high molecular mass (approximately 200 kDa and >200 kDa doublet) MMP-1 containing complexes. ProMMP-1 and MMP-1 containing complexes also occurred in wound fluid from venous stasis ulcers, but neither was detected in mastectomy fluid or in plasma. Levels of the proteinase inhibitor alpha2-macroglobulin in burn fluid and chronic ulcer wound fluid were almost as high as in plasma, and the high molecular mass MMP-1 containing complexes in burn fluid appeared to result from binding between alpha2-macroglobulin and activated MMP-1. These observations provide direct evidence that active MMP-1 in the fluid phase of the wound environment becomes complexed to alpha2-macroglobulin.

Acute Disease↗
PubMed1985

Biosynthesis and Intracellular Transport of 11S Globulin in Developing Pumpkin Cotyledons.

In vitro studies to explore the biosynthesis of 11S globulin developing cotyledons of pumpkin (Cucurbita sp.) demonstrated that 11S globulin is synthesized on membrane-bound polysomes. M(r) of the translation products (preproglobulin) synthesized by the poly(A)(+)-RNA isolated from developing cotyledons were determined to be 64,000 and 59,000, which are larger than those of the mature globulin subunit (62,000 and 57,000). Preproglobulin is then cotranslationally processed by cleavage of the signal peptide to produce proglobulin. In vivo pulse-chase experiments showed the sequential transformation of the single-chain proglobulin to mature globulin subunit (disulfide-linked doublet polypeptides) indicating posttranslational modification of the proglobulin.Subcellular fractionation of the pulse-chased intact cotyledons showed that the [(35)S]methionine label is detectable in proglobulin in rough endoplasmic reticulum shortly after the pulse label. With time, the labeled proteins move into other cellular fractions: proglobulin in the density = 1.24 grams per cubic centimeter fractions after 30 minutes and mature globulin subunit associated with protein bodies after 1 to 2 hours. The distribution of proglobulin in sucrose density gradients did not correspond with those of catalase (microbody marker) or fumarase (mitochondria marker). An accumulation of proglobulin occurred in the density = 1.24 grams per cubic centimeter fractions, whereas the mature globulin was scarcely detectable in this fraction. In contrast, proglobulin was not detected by immunochemical blotting analysis in the protein bodies prepared under the mild conditions from cotyledon protoplasts. The results suggest that the d = 1.24 grams per cubic centimeter fractions are engaged in the translocation of proglobulin into the protein bodies.

Journal Article↗
PubMed1994

H1TF2A, the large subunit of a heterodimeric, glutamine-rich CCAAT-binding transcription factor involved in histone H1 cell cycle regulation.

H1TF2 is a CCAAT transcription factor that binds to the histone H1 subtype-specific consensus sequence, which has previously been shown to be necessary for temporal regulation of histone H1 transcription during the cell cycle (F. La Bella, P. Gallinari, J. McKinney, and N. Heintz, Genes Dev. 3:1982-1990, 1989). In this study, we report that H1TF2 is a heteromeric CCAAT-binding protein composed of two polypeptide doublets of 33 and 34 kDa and 43 and 44 kDa that are not antigenically related. The 33- and 34-kDa species were not detected in our previous studies (P. Gallinari, F. La Bella, and N. Heintz, Mol. Cell. Biol. 9:1566-1575, 1989) because of technical problems in detection of these heavily glycosylated subunits. The cloning of H1TF2A, the large subunit of this factor, reveals it to be a glutamine-rich protein with extremely limited similarity to previously cloned CCAAT-binding proteins. A monospecific antiserum produced against bacterially synthesized H1TF2A was used to establish that HeLa cell H1TF2A is phosphorylated in vivo and that, in contrast to the H2b transcription factor Oct1 (S. B. Roberts, N. Segil, and N. Heintz, Science 253:1022-1026, 1991; N. Segil, S. B. Roberts, and N. Heintz, Cold Spring Harbor Symp. Quant. Biol. 56:285-292, 1991), no gross change in H1TF2A phosphorylation is evident during the cell cycle. Further immunoprecipitation studies demonstrated that H1TF2 is heterodimeric in the absence of DNA in vivo and identified several H1TF2-interacting proteins that may play a role in H1TF2 function in vivo.

Amino Acid Sequence↗
PubMed1996

Morphologic alterations in the cilia of a cat.

Based upon ultrastructural findings and computed tomography, a presumptive diagnosis of feline primary ciliary dyskinesia was made in a 2.5-year-old cat. The cat demonstrated morphologic alterations in the ultrastructure of oviductal cilia. In the oviduct, axonemal abnormalities were detected in 132 (20%) of 660 cross-sectioned cilia. The main ultrastructural ciliary defects were a lack of central microtubules, transposition of peripheral doublets to the center of the axoneme, supernumerary microtubules, and morphologic abnormalities of peripheral doublets. Computed tomography of the chest was consistent with early lesions of bronchiectasis, suggesting chronic stagnation of respiratory secretions, attributed to abnormal function of respiratory cilia. Specifically, the cranial branches of the cat's bronchi were wider and thicker than those of five healthy controls. Foci of pleural thickening and interstitial enlargement were also observed.

Animals↗
PubMed1986

Nuclear antigens recognized by antibodies present in liver disease sera.

Nuclear and nuclear matrix proteins of HeLa cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and subsequently transferred onto nitrocellulose. Antibodies present in sera of patients with primary biliary cirrhosis and autoimmune chronic active hepatitis reacted with some of the blotted proteins. The antibodies were mainly directed against chromatin-associated proteins and protein constituents of discrete RNP particles. In addition, antibodies found in autoimmune liver disease sera detected a hitherto undescribed nuclear protein of 54 kD, and a nuclear matrix protein of approximately 150 kD. Antibodies recognizing a nuclear 25 kD doublet apparently constituted a marker antibody for autoimmune liver disease. Those directed at the 17 kD centromere protein were associated with the primary biliary cirrhosis-related CREST syndrome, while those recognizing La antigen were related to cases of sicca syndrome associated with autoimmune liver diseases.

Antibodies, Antinuclear↗
PubMed1990

Ascorbyl free radical as a reliable indicator of free-radical-mediated myocardial ischemic and post-ischemic injury. A real-time continuous-flow ESR study.

The real-time kinetics of the release of ascorbyl free radicals in the coronary perfusate from isolated rat hearts submitted to an ischemia/reperfusion sequence has been achieved by continuous-flow ESR using high-speed acquisition techniques. Enhanced ESR detection of ascorbyl free radicals was obtained by addition of dimethyl sulfoxide (Me2SO), a strong cation chelator and oxidizing agent. A continuous-flow device allowed a direct monitoring of the ascorbyl free radical and/or ascorbate leakage in coronary perfusate by observation of the ascorbyl radical doublet (aH = 0.188 mT and g = 2.0054). 1. The results showed that ascorbyl free radical release occurred mainly during sequences of low-flow ischemia (90 min) coupled or not with 30 min of zero-flow ischemia followed by reperfusion (60 min). The kinetic profiles of ascorbyl-free-radical detection confirm in quantitative terms the expected correlation between the duration of the ischemic insult and the magnitude of ascorbate extracellular release upon reperfusion. There is indication that ascorbyl free radical depletion could be secondary to oxygen-derived-free-radical-induced cellular damage. 2. The amount of residual ascorbic acid was quantitated on myocardial tissue at the end of reperfusion using Me2SO as extracting solvent. Intense oxidation of ascorbate and chemical stabilization of the resulting free radical species provided by Me2SO allowed ESR measurement of a marked tissue ascorbate depletion related to the duration of ischemia. 3. Perfusion of superoxide dismutase during low-flow ischemia and the first 10 min of reperfusion greatly inhibited both extracellular release and endogenous ascorbate depletion. These results suggest that the ascorbate redox system constitutes a major protective mechanism against free-radical-induced myocardial injury. 4. The proposed direct ESR detection of ascorbyl free radicals in the coronary perfusates or in tissue extracts does not require extensive chemical preparation and conditioning of effluent or tissue samples. It provides an interesting straightforward alternative to the evaluation of detrimental free radical processes affecting the myocardium during ischemia and reperfusion.

Animals↗
PubMed2005

Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes.

An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the cross-linked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative "cross-linked peptides" (dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis(succinimidylsuccinate) derivate (D12-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H12 counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H12 form of the original cross-linked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H12 form of the original cross-linked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the cross-linked peptides after cleavage, has been shown to be a powerful tool for studies of multi-component protein complexes.

Algorithms↗
PubMed2004

Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase.

A key step in generation of the catalytically essential tyrosyl radical (Y122(*)) in protein R2 of Escherichia coli ribonucleotide reductase is electron transfer (ET) from the near-surface residue, tryptophan 48 (W48), to a (Fe(2)O(2))(4+) complex formed by addition of O(2) to the carboxylate-bridged diiron(II) cluster. Because this step is rapid, the (Fe(2)O(2))(4+) complex does not accumulate and, therefore, has not been characterized. The product of the ET step is a "diradical" intermediate state containing the well-characterized Fe(IV)Fe(III) cluster, X, and a W48 cation radical (W48(+)(*)). The latter may be reduced from solution to complete the two-step transfer of an electron to the buried diiron site. In this study, a (Fe(2)O(2))(4+) state that is probably the precursor to the X-W48(+)(*) diradical state in the reaction of the wild-type protein (R2-wt) has been characterized by exploitation of the observation that in R2 variants with W48 replaced with alanine (A), the otherwise disabled ET step can be mediated by indole compounds. Mixing of the Fe(II) complex of R2-W48A/Y122F with O(2) results in accumulation of an intermediate state that rapidly converts to X upon mixing with 3-methylindole (3-MI). The state comprises at least two species, of which each exhibits an apparent Mössbauer quadrupole doublet with parameters characteristic of high-spin Fe(III) ions. The isomer shifts of these complexes and absence of magnetic hyperfine coupling in their Mössbauer spectra suggest that both are antiferromagnetically coupled diiron(III) clusters. The fact that both rapidly convert to X upon treatment with a molecule (3-MI) shown in the preceding paper to mediate ET in W48A R2 variants indicates that they are more oxidized than X by one electron, which suggests that they have a bound peroxide equivalent. Their failure to exhibit either the long-wavelength absorption (at 650-750 nm) or Mössbauer doublet with high isomer shift (>0.6 mm/s) that are characteristic of the putatively mu-1,2-peroxo-bridged diiron(III) intermediates that have been detected in the reactions of methane monooxygenase (P or H(peroxo)) and variants of R2 with the D84E ligand substitution suggests that they have geometries and electronic structures different from those of the previously characterized complexes. Supporting this deduction, the peroxodiiron(III) complex that accumulates in R2-W48A/D84E is much less reactive toward 3-MI-mediated reduction than the (Fe(2)O(2))(4+) state in R2-W48A/Y122F. It is postulated that the new (Fe(2)O(2))(4+) state is either an early adduct in an orthogonal pathway for oxygen activation or, more likely, the successor to a (mu-1,2-peroxo)diiron(III) complex that is extremely fleeting in R2 proteins with the wild-type ligand set but longer lived in D84E-containing variants.

Electron Transport↗
PubMed1995

Characterization of a winged bean (Psophocarpus tetragonolobus) protein kinase with calmodulin-like domain: regulation by autophosphorylation.

A soluble protein kinase purified from winged bean (Psophocarpus tetragonolobus) shoots, has been assessed as a monomeric enzyme with an approximate M(r) of 60,000 in spite of the presence of two polypeptides of 61 and 58 kDa determined by SDS/PAGE. Immunoblot analyses using either of the two antisera raised individually against the polypeptides, detect both of them in purified preparations and a single larger polypeptide (62 kDa) in freshly prepared tissue homogenates, clearly indicating the likelihood of the doublet being formed from the larger one by proteolysis. Histone H1, syntide 2 and a synthetic myosin light chain-related peptide (MLC-peptide) have been identified as exogenous substrates of the enzyme. Complete Ca(2+)-dependence for substrate phosphorylation, a drastic inhibition of the reaction by a calmodulin (CaM) antagonist which can be partially reversed by a heterologous CaM and direct 45Ca(2+)-binding on blot, form compelling evidence in favour of a CaM-like domain of the enzyme. Both the polypeptides of the purified enzyme undergo intramolecular autophosphorylation on serine residue(s). Unlike the substrate phosphorylation reaction, autophosphorylation is Ca(2+)-independent and is not inhibited by the CaM antagonist. Down-regulation of substrate phosphorylation by auto-phosphorylation, and stimulation of the autophosphorylation by histone H1 and MLC-peptide, are novel regulatory features of the enzyme.

Amino Acid Sequence↗